MEMBRANE SPECIFIC MAPPING AND COLOCALIZATION OF MALARIAL AND HOST SKELETAL PROTEINS IN THE PLASMODIUM-FALCIPARUM-INFECTED ERYTHROCYTE BY DUAL-COLOR NEAR-FIELD SCANNING OPTICAL MICROSCOPY

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-functio n relationships, including elucidation of protein-protein interactions . We describe the use of a near field scanning optical microscope (NSO M) to simultaneously map and detect colocalized proteins within a cell , with superresolution. The system we elected to study was that of hum an red blood cells invaded by the human malaria parasite Plasmodium fa lciparum. During intraerythrocytic growth, the parasite expresses prot eins that are transported to the erythrocyte cell membrane. Associatio n of parasite proteins with host skeletal proteins leads to modificati on of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and para site proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studie s with laser scanning confocal microscopy provided lower resolution (3 10 nm) fluorescence maps of cross sections through the cell. Because t he two excitation colors shared the exact same near-field aperture, th e two fluorescence images were acquired in perfect, pixel-by-pixel reg istry, free from chromatic aberrations, which contaminate laser scanni ng confocal microscopy measurements. Colocalization studies of the pro tein pairs of mature parasite-infected erythrocyte surface antigen (ME SA) (parasite)/protein4.1 (host) and P. falciparum histidine rich prot ein (PfHRP1)(parasite)/protein4.1 (host) showed good real-space correl ation for the MESA/protein4.1 pair, but relatively poor correlation fo r the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in bi ological membranes. This method of detecting colocalization of protein s in cellular structures may have general applicability in many areas of current biological research.